Browsing by Author "Zhang, Sihui"
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- The ALS-inducing factors, TDP43A315T and SOD1G93A, directly affect and sensitize sensory neurons to stressVaughan, Sydney K.; Sutherland, Natalia M.; Zhang, Sihui; Hatzipetros, Theo; Vieira, Fernando; Valdez, Gregorio (Nature, 2018-11-08)There is increased recognition that sensory neurons located in dorsal root ganglia (DRG) are affected in amyotrophic lateral sclerosis (ALS). However, it remains unknown whether ALS-inducing factors, other than mutant superoxide dismutase 1 (SOD1G93A), directly affect sensory neurons. Here, we examined the effect of mutant TAR DNA-binding protein 1 (TDP43A315T) on sensory neurons in culture and in vivo. In parallel, we reevaluated sensory neurons expressing SOD1G93A. We found that cultured sensory neurons harboring either TDP43A315T or SOD1G93A grow neurites at a slower rate and elaborate fewer neuritic branches compared to control neurons. The presence of either ALS-causing mutant gene also sensitizes sensory neurons to vincristine, a microtubule inhibitor that causes axonal degeneration. Interestingly, these experiments revealed that cultured sensory neurons harboring TDP43A315T elaborate shorter and less complex neurites, and are more sensitive to vincristine compared to controls and to SOD1G93A expressing sensory neurons. Additionally, levels of two molecules involved in stress responses, ATF3 and PERK are significantly different between sensory neurons harboring TDP43A315T to those with SOD1G93A in vitro and in vivo. These findings demonstrate that sensory neurons are directly affected by two ALS-inducing factors, suggesting important roles for this neuronal subpopulation in ALS-related pathogenesis.
- Control of sex myoblast migration in C. elegansZhang, Sihui (Virginia Tech, 2013-08-01)Cell migration is critical in generating complex animal forms during development; misregulation of migration contributes to pathological conditions such as cancer metastasis. Thanks to its easily traceable cell lineages in a transparent body and a compact genome accessible to a wealth of genetic manipulations, the use of the nematode C. elegans as a model system has greatly advanced our understanding of mechanisms governing cell migration conserved through higher organisms. Among several migration processes in C. elegans, sex myoblast (SM) migration is an attractive system that has a simple and well-defined migratory route along the ventral side from the posterior to the precise center of the gonad. A multitude of guidance mechanisms control SM migration, many of which are likely to be conserved in other migratory processes. Similar to vertebrate systems, C. elegans uses Rho family small GTPases to regulate the engine of cell motility, the actin cytoskeleton, in response to guidance cues. The differential utilizations of Rho GTPases in distinct processes in vivo remain a central question in the study of Rho GTPases. I investigated how Rho GTPases regulate different aspects of SM migration, and found that Cdc-42/CDC42 functions in the anteroposterior migration, whereas MIG-2/RhoG and CED-10/Rac1 control ventral restriction independently of FGF and SLIT/Robo signaling. The relative difficulty in perturbing SM migration using constitutively active Rho GTPases compared to other migration processes illustrates the robustness of the mechanisms that control SM migration. On a technical aspect, I established a nematode larval cell culture system that allows access to postembryonic cells. Compared to the flourishing genetic researches in C. elegans, there are few studies of molecules that also extend to the subcellular level in postembryonic development, mainly due to the lack of a larval cell culture system. I developed a novel method combining SDS-DTT presensitization of larval cuticles and subsequent pronase E digestion. My method efficiently isolates both low- and high-abundance cell types from all larval stages. This technical advance will not only facilitate studies such as regulation of actin dynamics with high-resolution microscopy, but is beginning to be used by researchers to tackle cell-type specific questions through profiling methods as gene expression analysis.
- Isolation and Culture of Larval Cells from C. elegansZhang, Sihui; Banerjee, Diya; Kuhn, Jeffrey R. (PLOS, 2011-04-29)Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×104 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.